An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells

Abstract

Background: Virus-binding activity is one of the important functions of fibronectin (FN). It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported.

Methods: We employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN) was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN in vivo became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA.

Results: The protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase). The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase) cleaved FN at four sites, R259, R1005, K1557 and R2039, among which the digested fragments at R259, K1557 and R2039 were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.

Conclusion: The earthworm fibronectinase (EFNase) cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.

Figure 1

Purification of EFNase. SDS-PAGE of EFNase purified from Eisenia fetida. Lane 1 is the protein molecule markers; lane 2 represents EFNase.

Figure 2

Digestion of human serum in the presence of EFNase. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at different time intervals during the degradation and then aliquots were taken for SDS-PAGE (panel A). Under the same conditions, serum in the absence of EFNase was used as negative control (panel B). Panel C was the comparison of the hydrolytic activity of EFNase and trypsin by densitomitric scanning analysis of Figure 2A and Additional file 1B.

Figure 3

The sequences of the matched peptides. Match to fibronectin 1, Homo sapiens, Q60FE4, GenBank.

Figure 4

The further proof of 'fibronectin' in serum by western blotting. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at 37°C for 120 min, and then aliquots were taken for SDS-PAGE and western blotting (panel A). EFNase was also added to serum and aliquots were taken for SDS-PAGE and western blotting at different time intervals during the degradation (panel B).

Figure 5

Changes in fibronectin concentrations after EFNase oral administrated to rats. EFNase was oral administrated to rats at a dose 2000 mg/kg. Panel A was a typical western blotting picture of the serum. Lane 1 showed the serum before oral administration of EFNase as control; Lanes 2 through 9 were serum after oral administration of EFNase at time as indicated. Panel B was the FN concentrations determined by ELISA (Mean ± SD, n = 6). * P < 0.05, **P < 0.01 versus the FN concentration before oral administration of EFNase.

Figure 6

Digestion of fibronectin by EFNase. EFNase (final concentrations as indicated) was incubated with FN at 37°C and aliquots were taken at different time intervals for reducing SDS-PAGE during the degradation (panel A). Under the same conditions, FN in the presence of EFNase was analyzed by non-reducing SDS-PAGE, compared with reducing SDS-PAGE. Lanes 2 through 7 were non-reducing SDS-PAGE and lanes 8 to 9 were reducing SDS-PAGE (panel B).

Figure 7

The main function domain of fibronectin digested by EFNase. The functional domains of FN digested by EFNase were shown in this panel. Ligands interaction regions and cleavage sites are shown (discussed in text). Variably spliced extradomain A (A) and extradomain B (B) modules and variable sequence (V) are shown in the subunit of the FN dimer.

Figure 8

Effect of EFNase on HBeAg and HBsAg secreted HepG2.2.15 cells and the cell viability. HBsAg and HBeAg in cell culture in the presence of EFNase (panel A) and lamivudine (panel B) were analyzed by ELISA. Cell viabilities treated with EFNase (panel C) and lamivudine (panel D) were determined using CCK-8 kits. The data represent means ± SD (n = 3).